Method for specific binding assays using a releasable ligand

Abstract

Immunoassays and DNA probe assays utilizing a non-immune, reversible binding displacement system are provided. In the assay, a releasable ligand, a binding partner for the releasable ligand, an analyte of interest, an analytically detectable (reporter) group, and at least one binding partner for the analyte, are first attached to an insoluble phase so as to form reporter-labeled complex bound to an insoluble phase, followed by the addition of a displacer ligand which displaces the releasable ligand along with some portion of the reporter-labeled complex, so that the released reporter is analytically detectable in a free liquid medium and can be related to the concentration of analyte in the sample.

What is claimed is: 
     
       1. A non-competitive specific binding assay for an analyte comprising the steps of: A. preparing an immobilized sandwich structure consisting essentially of: 1. a solid support having a first binding partner attached thereto wherein the first binding partner is selected from the group consisting of nucleic acid and antibody;   2. a second binder partner:analyte complex; and   3. a releasable ligand, wherein said releasable ligand is attached through a temporary bond to a third binding partner having detectable reporter thereon and through a covalent bond to the second binding partner of the second binding partner:analyte complex;      by contacting said solid support having a first binding partner capable of binding to the analyte attached thereto with: 1. liquid sample suspected of containing the analyte;   2. releasable ligand attached through a covalent bond to the second binding partner which is capable of binding to the analyte; and   3. the third binding partner capable of binding to the releasable ligand and having detectable reporter thereon;     B. separating the immobilized sandwich structure from soluble components;   C. breaking the temporary bond by: 1. adding an excess of a displacer ligand relative to the releasable ligand; or   2. adding a displacer ligand wherein the affinity of the displacer ligand to the third binding partner is greater than the affinity of the releasable ligand to the third binding partner;     D. measuring the detectable reporter in solution; and   E. relating the measured detectable reporter to the amount of analyte in the liquid sample.   
     
     
       2. The non-competitive specific binding assay of claim 1 wherein the releasable ligand is dethiobiotin, the third binding partner is streptavidin and the displacer ligand is biotin. 
     
     
       3. The non-competitive specific binding assay of claim 1 wherein the releasable ligand is streptavidin, the third binding partner is dethiobiotin and the displacer ligand is biotin. 
     
     
       4. The non-competitive specific binding assay of claim 1 wherein the releasable ligand is attached to the second binding partner through a hydrophilic spacer selected from the group consisting of alpha, omega-amino acids and derivatives thereof. 
     
     
       5. A non-competitive specific binding assay for an analyte comprising the steps of: A. preparing an immobilized sandwich structure consisting essentially of: 1. a solid support having a first binding partner attached thereto wherein the first binding partner is selected from the group consisting of streptavidin, avidin and succinylated avidin;   2. a releasable ligand; wherein said releasable ligand is attached through a temporary bond to the first binding partner; and   3. a second binding partner:analyte complex, wherein the second binding partner of the second binding partner:analyte complex is attached through a covalent bond to the releasable ligand and the analyte of the second binding partner: analyte complex is attached to a third binding partner having a detectable reporter thereon;      by contacting said solid support having a first binding partner capable of binding to the releasable ligand attached thereto with: 1. liquid sample suspected of containing the analyte;   2. releasable ligand attached through a covalent bond to the second binding partner which is capable of binding to the analyte; and     
     
     
       3. the third binding partner capable of binding to the analyte and having a detectable reporter thereon; B. separating the immobilized sandwich structure from soluble components;   C. breaking the temporary bond by: 1. adding an excess of displacer ligand relative to the releasable ligand; or   2. adding a displacer ligand wherein the affinity of the displacer ligand to the first binding partner is greater than the affinity of the releasable ligand to the first binding partner, wherein the displacer ligand is a ligand that is capable of binding said first binding partner;     D. measuring the detectable reporter in solution; and   E. relating the measured detectable reporter to the amount of analyte in the liquid sample.   
     
     
       6. The non-competitive specific binding assay of claim 5 wherein the releasable ligand is selected from the group consisting of biotin, dethiobiotin, iminobiotin and functionalized azo dye and the displacer ligand is selected from the group consisting of biotin and dethiobiotin. 
     
     
       7. The non-competitive specific binding assay of claim 5 wherein the releasable ligand is attached to the second binding partner through a hydrophilic spacer selected from the group consisting of alpha, omega-amino acids and derivatives thereof. 
     
     
       8. A non-competitive specific binding assay for the analyte comprising the steps of: A. preparing an immobilized sandwich structure consisting essentially of: 1. a solid support having a first binding partner attached thereto wherein the first binding partner is selected from the group consisting of nucleic acid and antibody;   2. an analyte;   3. a releasable ligand, wherein said releasable ligand is attached through a temporary bond to a second binding partner having detectable reporter thereon and through a covalent bond to the analyte;      by contacting said solid support having a first binding partner capable of binding to the analyte attached thereto with:   
     
     
       1. liquid sample suspected of containing the analyte which has been reacted with a releasable ligand which has been activated so that it is capable of covalently binding to the analyte; and 2. the second binding partner capable of binding to the releasable ligand and having detectable reporter thereon;   B. separating the immobilized sandwich structure from soluble components;   C. breaking the temporary bond by: 2. adding an excess of a displacer ligand relative to the releasable ligand; or   2. adding a displacer ligand wherein the affinity of the displacer ligand to the second binding partner is greater than the affinity of the releasable ligand to the second binding partner;     D. measuring the detectable reporter in solution; and   E. relating the measured detectable reporter to the amount of analyte in the liquid sample.   
     
     
       9. The non-competitive specific binding assay of claim 8 wherein the releasable ligand is selected from the group consisting of biotin, dethiobiotin, iminobiotin and functionalized azo dye; the second binding partner is selected from the group consisting of streptavidin, succinylated avidin and avidin; and the displacer ligand is selected from the group consisting of biotin, dethiobiotin and streptavidin. 
     
     
       10. The non-competitive specific binding assay of claim 8 wherein the releasable ligand is attached to the analyte through a hydrophilic spacer selected from the group consisting of alpha, omega-amino acids and derivatives thereof. 
     
     
       11. A competitive specific binding assay for an analyte comprising the steps of: A. immobilizing on a solid support an analyte;   B. forming an immobilized reporter-labeled complex by contacting the product of step A with a mixture containing reporter-labeled complex prepared by combining:   
     
     
       1. a liquid sample containing an analyte; 2. a known quantity of a first binding partner attached through a covalent bond to a releasable ligand, said first binding partner being capable of binding to the analyte; and   3. a second binding partner having detectable reporter thereon, wherein said second binding partner is capable of being attached through a temporary bond to the releasable ligand;   C. separating the immobilized reporter-labeled complex from the soluble components;   D. breaking the temporary bond by: 1. adding an excess of a displacer ligand relative to the releasable ligand; or   2. adding a displacer ligand wherein the affinity of the displacer ligand to the second binding partner is greater then the affinity of the releasable ligand to the second binding partner;     E. measuring the reporter in solution; and   F. relating the measured detectable reporter to the amount of analyte in the liquid sample.   
     
     
       12. The competitive specific binding assay of claim 11 wherein the releasable ligand is selected from the group consisting of biotin, dethiobiotin, iminobiotin and functionalized azo dye; the second binding partner is selected from the group consisting of streptavidin, succinylated avidin and avidin; and the displacer ligand is selected from the group consisting of biotin and dethiobiotin. 
     
     
       13. The competitive specific binding assay of claim 10 wherein the releasable ligand is capable of being attached to the first binding partner through a hydrophilic spacer selected from the group consisting of alpha omega-amino acids and derivatives thereof. 
     
     
       14. A competitive specific binding assay for an analyte comprising the steps of: A. immobilizing on a solid support a first binding partner capable of binding to a releasable ligand through a temporary bond;   B. forming an immobilized reporter-labeled complex by contacting the product of step A with a mixture containing reporter-labeled complex prepared by combining:   
     
     
       1. a liquid sample containing the analyte; 2. a known quantity of a releasable ligand: analyte conjugate wherein the releasable ligand is capable of being attached through a temporary bond to the first binding partner on the solid support; and   3. a known quantity of a second binding partner capable of binding with the analyte and having detectable reporter thereon;   C. separating the immobilized reporter-labeled complex from soluble components;   D. breaking the temporary bond by: 1. adding an excess of a displacer ligand relative to the releasable ligand; or   2. adding a displacer ligand wherein the affinity of the displacer ligand to the first binding partner is greater than the affinity of the releasable ligand to the first binding partner, wherein the displacer ligand is a ligand that is capable of binding said first binding partner;     E. measuring the reporter in solution; and   F. relating the measured detectable reporter to the amount of analyte in the liquid sample.   
     
     
       15. The competitive specific binding assay of claim 14 wherein the first binding partner is selected from the group consisting of streptavidin, succinylated avidin and avidin; the releasable ligand is selected from the group consisting of biotin, dethiobiotin, iminobiotin and functionalized azo dye; and the displacer ligand is selected from the group consisting of biotin, dethiobiotin and streptavidin. 
     
     
       16. The competitive specific binding assay of claim 14 wherein the releasable ligand is attached to the analyte through a hydrophilic spacer selected from the group consisting of alpha, omega-amino acids and derivatives thereof. 
     
     
       17. A non-competitive specific binding assay for analyte comprising the steps of: A. preparing an immobilized sandwich structure consisting essentially of: 1. a solid support having a first binding partner attached thereto wherein the first binding partner is selected from the group consisting of nucleic acid and antibody;   2. a second binding partner:analyte complex;     
     
     
       3. a first releasable ligand, wherein said first releasable ligand is attached through a first temporary bond to a third binding partner and through a covalent bond to the second binding partner of the second binding partner; analyte complex; and 4. a second releasable ligand having a detectable reporter thereon, wherein said second releasable ligand is attached through a second temporary bond to the third binding partner;    by contacting said solid support having a first binding partner capable of binding to the analyte attached thereto with: 1. liquid sample suspected of containing the analyte;   2. first releasable ligand attached through a covalent bond to the second binding partner;   3. the third binding partner capable of binding to the first releasable ligand; and   4. the second releasable ligand capable of binding to the third binding partner and having a detectable reporter thereon;     B. separating the immobilized sandwich structure from soluble components;   C. breaking the first and second temporary bonds by: 1. adding an excess of a displacer ligand relative to the first and second releasable ligand; or   2. adding a displacer ligand wherein the affinity of the displacer ligand to the third binding partner is greater than the affinity of the first and second releasable ligands to the third binding partner; and     D. measuring the detectable reporter in solution; and   E. relating the measured detectable reporter to the amount of analyte in the liquid sample.   
     
     
       18. The non-competitive specific binding assay of claim 17 wherein the first releasable ligand is attached to the second binding partner through a hydrophilic spacer selected from the group consisting of alpha omega-amino acids and derivatives thereof. 
     
     
       19. A competitive specific binding assay for an analyte comprising the steps of: A. immobilizing on a solid support an analyte;   B. forming an immobilized reporter labeled complex by contacting the product of step A with a mixture containing reporter-labeled complex prepared by combining:   
     
     
       1. a liquid sample containing the analyte; 2. a known quantity of a first binding partner attached through a covalent bond to a second binding partner, said first binding partner being capable of binding to the analyte; and   3. a releasable ligand having a detectable reporter thereon, wherein the second binding partner is capable of being attached through a temporary bond to the releasable ligand;   C. separating the immobilized reporter-labeled complex from the soluble components;   D. breading the temporary bond by: 1. adding an excess of a displacer ligand relative to the releasable ligand; or   2. adding a displacer ligand wherein the affinity of the displacer ligand to the first binding partner is greater than the affinity of the releasable ligand to the first binding partner; and     E. measuring the reporter in solution; and   F. relating the measured detectable reporter to the amount of analyte in the liquid sample.